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1.
Vaccine ; 19(30): 4143-52, 2001 Jul 20.
Article in English | MEDLINE | ID: mdl-11457539

ABSTRACT

Human respiratory syncytial virus (hRSV) is a major pathogen responsible for bronchiolitis and severe pulmonary disease in very young children, immunodeficient patients and the elderly. BBG2Na, a recombinant chimeric protein produced in Escherichia coli, is a promising subunit vaccine candidate against this respiratory pathogen, composed of G2Na, the central domain of RSV G glycoprotein, and BB, an albumin binding domain of streptococcal protein G. BBG2Na has a basic isoelectric point (pI 9.3) and as expected, is strongly adsorbed by aluminium phosphate (AP). Surprisingly, BBG2Na is also strongly adsorbed by aluminium hydroxide (AH), which normally binds molecules with acidic isoelectric points. This behaviour was unexpected according to the well established adsorption model of Hem and co-workers. Our observations may be explained by the bipolar two-domain structure of the BBG2Na chimera which is not reflected by the global basic isoelectric point of the whole protein: the BB domain has an acidic isoelectric point (pI 5.5) and the G2Na domain a highly basic one (pI 10.0). Importantly, formulation in either aluminium salt resulted in equally high immunogenicity and protective efficacy against RSV in mice. From a physicochemical point of view, this unique property of BBG2Na makes it eminently suitable for combination to either paediatric or elderly multivalent AH- or AP-containing vaccines already in the market or in development.


Subject(s)
Adjuvants, Immunologic/administration & dosage , Aluminum Compounds/administration & dosage , Aluminum Hydroxide/administration & dosage , Phosphates/administration & dosage , Respiratory Syncytial Virus, Human/immunology , Vaccines, Synthetic/immunology , Viral Vaccines/immunology , Adsorption , Amino Acid Sequence , Animals , Buffers , Ethylene Glycol/pharmacology , Hydrogen-Ion Concentration , Mice , Mice, Inbred BALB C , Molecular Sequence Data , Vaccines, Subunit/immunology
2.
J Immunol Methods ; 251(1-2): 151-9, 2001 May 01.
Article in English | MEDLINE | ID: mdl-11292490

ABSTRACT

We have developed and validated a process-specific immunoligand assay based on the Threshold system for the quantification of residual host cell proteins (HCPs) in a recombinant subunit vaccine candidate against the human respiratory syncytial virus (hRSV). The industrial process of this vaccine produced in Escherichia coli, involved five chromatography steps for the production of clinical-grade batches. The clearance of non-product-related protein throughout the purification process was documented by the evaluation of the HCP content in the chromatographic fractions at each step of the downstream processing. The assay had a detection limit of 0.5 ng/ml of HCP equivalent to 10 parts per million (ppm). The quantification limit was 1.3 ng/ml of HCP, giving a sensitivity range of the assay of 10 to 30 ppm. To our knowledge, this is the first sensitive HCP assay reported for a vaccine.


Subject(s)
Immunoassay/methods , Respiratory Syncytial Viruses/genetics , Respiratory Syncytial Viruses/immunology , Viral Vaccines/analysis , Antibodies, Bacterial , Antibody Specificity , Bacterial Proteins/analysis , Bacterial Proteins/immunology , Drug Contamination , Escherichia coli/genetics , Escherichia coli/immunology , Humans , Immunoassay/statistics & numerical data , In Vitro Techniques , Sensitivity and Specificity , Vaccines, Subunit/analysis , Vaccines, Synthetic/analysis
3.
Peptides ; 18(2): 293-300, 1997.
Article in English | MEDLINE | ID: mdl-9149303

ABSTRACT

In order to investigate the putative physiological role of the in vivo release of hemorphins from hemoglobin in tissues, an immunological approach was developed. Specific and sensitive antiserum were raised against the C-part of the V-V-hemorphin-7. The antisera recognized to the same extent the related hemorphins V-V-hemorphin-7 and L-V-V-hemorphin-7. The validity of our immunological approach was analyzed by studying the in vitro release of hemorphin from hemoglobin by cathepsin D and compared to the pepsin hydrolysis. These two enzymes led to the release of these same products suggesting that cathepsin D acted as an accurate pepsin-like enzyme. Moreover, considering the poor sensitivity of the available methods of detection for the in vitro Cathepsin D activity, our specific and sensitive V-V-hemorphin-7 radioimmunoassay seems to be a useful alternative assay for this enzymatic activity.


Subject(s)
Cathepsin D/metabolism , Hemoglobins/metabolism , Peptide Fragments/analysis , Peptide Fragments/chemistry , Amino Acid Sequence , Animals , Antibody Specificity , Cattle , Chromatography, High Pressure Liquid , Cross Reactions , Hydrolysis , Immune Sera , Pepsin A/metabolism , Peptide Fragments/metabolism , Radioimmunoassay , Sensitivity and Specificity
4.
FEBS Lett ; 382(1-2): 37-42, 1996 Mar 11.
Article in English | MEDLINE | ID: mdl-8612760

ABSTRACT

Bovine globin has been incubated with mice peritoneal macrophages in order to study its hydrolysis by lysosomal enzymes, among which chiefly cathepsin D. Analysis of resulting peptides, by reversed-phase high-performance liquid chromatography (RP-HPLC), showed the release of a bioactive peptide, VV-hemorphin-7. When a carboxyl proteinase inhibitor such as pepstatin A was added, no hemorphin was generated. Our results clearly demonstrated that VV-hemorphin-7 generation was principally due to cathepsin D. This study allowed us to hypothesize a possible pathway for in vivo hemorphins appearance from globin catabolism by macrophages.


Subject(s)
Globins/metabolism , Hemoglobins/metabolism , Macrophages, Peritoneal/metabolism , Peptide Fragments/metabolism , Amino Acid Sequence , Amino Acids/analysis , Animals , Cathepsin D/metabolism , Cattle , Cells, Cultured , Female , Hemoglobins/chemistry , Hydrolysis , Leupeptins/pharmacology , Lysosomes/enzymology , Mice , Molecular Sequence Data , Pepstatins/pharmacology , Peptide Fragments/chemistry , Protease Inhibitors/pharmacology , Specific Pathogen-Free Organisms
5.
Neuropeptides ; 30(1): 1-5, 1996 Feb.
Article in English | MEDLINE | ID: mdl-8868292

ABSTRACT

Bovine globin has been hydrolysed by pepsin to different degrees of hydrolysis. Analysis of the hydrolysates, by reversed-phase high-performance liquid chromatography (RP-HPLC), shows the release of LVV- and VV-hemorphin-7. LVV-hemorphin-7 was the first generated, at a degree of hydrolysis (DH), as low as 4%. In contrast, VV-hemorphin-7 was produced later. Our study clearly shows that VV-hemorphin-7 is issued directly from LVV-hemorphin-7, since this later completely disappeared during hydrolysis. This work allows us to suggest a possible pathway for in vivo hemorphins appearance.


Subject(s)
Hemoglobins/chemistry , Peptide Fragments/chemistry , Amino Acids/analysis , Animals , Cattle , Chromatography, High Pressure Liquid , Globins/chemistry , Hydrolysis , Kinetics , Spectrophotometry, Ultraviolet
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